Methods for therapy of neurodegenerative disease of the brain

ABSTRACT

A specific clinical protocol for use toward therapy of defective, diseased and damaged neurons in the mammalian brain, of particular usefulness for treatment of neurodegenerative conditions such as Parkinson&#39;s disease and Alzheimer&#39;s disease. The protocol is practiced by delivering a definite concentration of recombinant neurotrophin, such as glial cell-derived neurotrophic factor), into a targeted region of the brain (such as the substantia nigra) using a lentiviral expression vector. The neurotrophin is delivered to, or within close proximity of, identified defective, diseased or damaged brain cells. The concentration of neurotrophin delivered as part of a neurotrophic composition varies from 10 10  to 10 15  neurotrophin encoding viral particles/ml of composition fluid. Each delivery site receives from 2.5 μl to 25 μl of neurotrophic composition, delivered slowly, as in over a period of time ranging upwards of 10 minutes/delivery site. Each delivery site is at, or within 500 μm of, a targeted cell, and no more than about 10 mm from another delivery site. The method stimulates growth of targeted neurons, and reversal of functional deficits associated with the neurodegenerative disease being treated.

RELATED U.S. PATENT APPLICATIONS

[0001] This is a continuation-in-part of, and claims the priority of,U.S. patent application, Ser. No. 09/620,174, filed on Jul. 19, 2000,still pending, which in turns claims the priority of 09/060,543, filedon Apr. 15, 1998, still pending.

FIELD OF THE INVENTION

[0002] The invention relates to methods for treatment ofneurodegenerative disease and methods for delivery of therapeuticneurotrophins into the mammalian brain.

HISTORY OF THE RELATED ART

[0003] Neurotrophins play a physiological role in the development andregulation of neurons in mammals. In adults, basal forebrain cholinergicneurons, motor neurons and sensory neurons of the CNS retainresponsiveness to neurotrophic factors and can regenerate after loss ordamage in their presence. For this reason, neurotrophins are consideredto have great promise as drugs for the treatment of neurodegenerativeconditions such as Alzheimer's Disease (AD), Parkinson's Disease (PD),amyotrophic lateral sclerosis (ALS), peripheral sensory neuropathies andspinal cord injuries.

[0004] Direct delivery of neurotrophins through infusion into theneurocompromised brain has been met with limited success and, in oneinstance, actually worsened the condition being treated (Kordower, etal., Ann. Neurol., 46:419-424, 1999 [symptoms of PD worsened followinginfusion of glial cell-derived neurotrophic factor]). In contrast, invivo transduction of CNS cells with a neurotrophin encoding expressionvector holds tremendous promise as a more broadly applicable method oftreating and preventing neurodegeneration. Ideally, the vector utilizedto deliver the neurotrophin will display at least moderate levels oftransduction efficiency, while producing minimal toxicity.

SUMMARY OF THE INVENTION

[0005] The invention provides a lentiviral-based, clinically usefulsystem and protocol for delivery of recombinant neurotrophins into themammalian brain. The invention is particularly useful in treatingneurodegenerative conditions in primates, in whom neurotrophinsdelivered according to the invention stimulate growth of neurons andrecovery of neurological function.

[0006] More specifically, the invention consists of methods forintraparenchymal delivery of neurotrophins to defective, diseased ordamaged cells in the mammalian brain using a lentiviral expressionvector. In one aspect, the invention provides a specific protocol foruse in genetically modifying target neurons (“target cells”) to producea therapeutic neurotrophin; e.g., in the substantia nigra or basalforebrain. The genetic modification of target cells is achieved by invivo transfection of neurons targeted for treatment, or by transfectionof cells neighboring these target neurons (neurons or glia), with arecombinant expression vector for expression of the desired neurotrophinin situ.

[0007] The location for delivery of individual unit dosages ofneurotrophin into the brain is selected for proximity to previouslyidentified defective, diseased or damaged target cells in the brain. Tointensify exposure of such target cells to the endogenous growthfactors, each delivery site is situated no more than about 500 μm from atargeted cell and no more than about 10 mm from another delivery site.The total number of sites chosen for delivery of each unit dosage ofneurotrophin will vary with the size of the region to be treated.

[0008] Optimally, for delivery of neurotrophin using the lentiviralexpression vector, each unit dosage of neurotrophin will comprise 2.5 to25 μl of an expression vector composition, wherein the compositionincludes a viral expression vector in a pharmaceutically acceptablefluid (“neurotrophic composition”) and provides from 10¹⁰ up to 10¹⁵ NGFexpressing viral particles per ml of neurotrophic composition.

[0009] This lentiviral based protocol for neurotrophin delivery achievesa high level of transduction efficiency, with minimal toxicity, toproduce a therapeutic or preventative effect in the primate brain.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1. (A) Dense GDNF immunoreactivity within the head of thecaudate nucleus and putamen in a lenti-GDNF-treated aged monkey. (B) Incontrast, no GDNF immunoreactivity was observed in these regions in alenti-Gal-treated animal. IC, internal capsule. (C) Dense GDNFimmunoreactivity was observed within the midbrain of alenti-GDNF-treated animal. (D) GDNF immunoreactivity within theforebrain of a lenti-GDNF-treated monkey. The staining within theputamen (Pt) is from an injection site. The staining within bothsegments of the globus pallidus (GPe and GPi) is the result ofanterograde transport. (E) Anterogradely transported GDNF was also seenin the substantia nigra pars reticulata. Note that the holes in thetissue sections were made post mortem for HPLC analysis. Asterisk in (E)represents a lenti-GDNF injection site (CP, cerebral peduncle). Scalebar in (D) represents 1600 μm for panels A, B, and D; 1150 μm for panelC, and 800 μm for panel E.

[0011]FIG. 2. PET scan data evaluating the influence of lenti-GDNF on FDuptake in (A and B) intact aged monkeys and (C and D) young adultMPTP-treated monkeys. (A) FD uptake did not change from baseline to 3months after lentivirus injection in lenti-Gal-treated aged monkeys. (B)In contrast, lenti-GDNF injections manifested increased FD uptake on theside of GDNF expression relative to preoperative levels in aged monkeys.Ki values (per minute) for the striatum are as follows: (left side)lenti-Gal preoperative 0.0068±0.0001, lenti-Gal postoperative0.0062±0.0002; (right side) lenti-Gal preoperative 0.0068±0.0002,lenti-Gal postoperative 0.0065±0.0001; (left side) lenti-GDNFpreoperative 0.0072±0.0005, lenti-GDNF postoperative 0.0068±0.0003;(right side) lenti-GDNF preoperative 0.0076±0.0004, lenti-GDNFpostoperative 0.0081±0.0003. (C) After MPTP lesions, a comprehensiveloss of FD uptake was seen within the right striatum oflenti-Gal-treated young adult monkeys. (D) In contrast, FD uptake wasenhanced in lenti-GDNF-treated monkeys. Ki values (per minute) for thestriatum are as follows: lenti-Gal left, 0.0091±0.0004; lenti-Gal right,0.0017±0.0005; lenti-GDNF left, 0.0084±0.0004; lenti-GDNF right,0.0056±0.0018.

[0012]FIG. 3. (A) Section stained for TH immunoreactivity through theanterior commissure illustrating the increase in TH immunoreactivitywithin the right caudate nucleus and putamen after lenti-GDNF deliveryto aged monkeys. (B) Symmetrical and less intense staining for THimmunoreactivity in a monkey injected with lenti-Gal. (C) There weregreater numbers and larger TH-immunoreactive neurons within thesubstantia nigra of a lenti-GDNF-treated animal relative to (D) alenti-Gal-treated monkey. (E) Lenti-GDNF-treated aged monkeys displayedincreased TH mRNA relative to (F) lenti-Gal-treated monkeys in the SN.Scale bar in (F) represents 4500 μm for panels 250 μm for panels (C) and(D) and 100 μm for panels (E) and (F).

[0013]FIG. 4. (A through F) Plots of quantitative data illustratingenhanced nigrostriatal function in lenti-GDNF-treated aged monkeys.Solid bars denote lenti-Gal-treated monkeys; hatched bars indicatelenti-GDNF-treated monkeys. GDNF expression was limited to the rightstriatum and nigra. **P<0.01; ***P<0.001.

[0014]FIG. 5. After MPTP-treatment, lenti-GDNF-injected monkeysdisplayed functional improvement on (A) the clinical rating scale and(B) the hand-reach task. All tests were performed 3 weeks per month [see(15)]. On the clinical rating scale, monkeys were matched into groupsbased upon the post-MPTP score. For the hand-reach task, each symbolrepresents the mean of three sessions per week for the left hand.Monkeys were not tested on this task during the week between MPTP andlentivirus injection. *P<0.05 relative to lenti-Gal.

[0015]FIG. 6. (A and B) Low-power dark-field photomicrographs throughthe right striatum of TH-immunostained sections of MPTP-treated monkeystreated with (A) lenti-Gal or (B) lenti-GDNF. (A) There was acomprehensive loss of TH immunoreactivity in the caudate and putamen oflenti-Gal-treated animal. In contrast, near normal level of THimmunoreactivity is seen in lenti-GDNF-treated animals. Low-power (C andD) and medium-power (E and F) photomicrographs of TH-immunostainedsection through the substantia nigra of animals treated with lenti-Gal(C and E) and lenti-GDNF (D and F). Note the loss of TH-immunoreactiveneurons in the lenti-Gal-treated animals on the side of theMPTP-injection. TH-immunoreactive sprouting fibers, as well as asupranormal number of TH-immunoreactive nigral perikarya are seen inlenti-GDNF-treated animals on the side of the MPTP injection. (G and H)Bright-field low-power photomicrographs of a TH-immunostained sectionfrom a lenti-GDNF-treated monkey. (G) Note the normal TH-immunoreactivefiber density through the globus pallidus on the intact side that wasnot treated with lenti-GDNF. (H) In contrast, an enhanced network ofTH-immunoreactive fibers is seen on the side treated with both MPTP andlenti-GDNF. Scale bar in (G) represents the following magnifications:(A), (B), (C), and (D) at 3500 μm; (E), (F), (G), and (H) at 1150 μm.

[0016]FIG. 7. (A through D) Quantification of lenti-GDNF's trophiceffects on nigral neuronal number, volume, TH mRNA and striatal THimmunoreactivity in MPTP-treated monkeys. ***P<0.001 significantdecreases relative to intact side; ttt denotes significant increasesrelative to the intact side.

DETAILED DESCRIPTION OF THE INVENTION

[0017] I. Target Tissues for Treatment of Neurodegenerative DisordersAccording to the Invention

[0018] The invention identifies and defines the required parameters of amethod for successful regeneration of neurons in the brain withneurotrophins, especially the neurons whose loss is associated withneurodegenerative conditions with impairment of cognition such as AD.

[0019] The first method parameter defined by the invention is selectionof a suitable target tissue. A region of the brain is selected for itsretained responsiveness to neurotrophic factors. In humans, CNS neuronswhich retain responsiveness to neurotrophic factors into adulthoodinclude the cholinergic basal forebrain neurons, dopaminergic neurons ofthe substantia nigra, the entorhinal cortical neurons, the thalamicneurons, the locus coeruleus neurons, the spinal sensory neurons and thespinal motor neurons.

[0020] In normal subjects, neurotrophins prevent sympathetic and sensoryneuronal death during development and prevents cholinergic neuronaldegeneration in adult rats and primates (Tuszynski, et al., GeneTherapy, 3:305-314 (1996)). The resulting loss of functioning neurons inthis region of the basal forebrain is believed to be causatively linkedto the cognitive decline experienced by subjects suffering fromneurodegenerative conditions such as AD. Similarly, loss offunctionality in dopaminergic neurons of the substantia nigra iscausatively associated with the onset of PD.

[0021] Treatment of the targeted region of the brain with vectorcomposition at upwards of 10 separate in vivo gene vector delivery sitesis desirable. Importantly, specific gene delivery sites are selected soas to cluster in an area of neuronal loss. Such areas may be identifiedclinically using a number of known techniques, including magneticresonance imaging (MRI) and biopsy. In humans, non-invasive, in vivoimaging methods such as MRI will be preferred. Once areas of neuronalloss are identified, delivery sites are selected for stereotaxicdistribution so each unit dosage of NGF is delivered into the brain at,or within 500 μm from, a targeted cell, and no more than about 10 mmfrom another delivery site.

[0022] II. Materials for Use in Practicing the Invention

[0023] Materials useful in the methods of the invention include in vivocompatible recombinant expression vectors, packaging cell lines, helpercell lines, synthetic in vivo gene therapy vectors, regulatable geneexpression systems, encapsulation materials, pharmaceutically acceptablecarriers and polynucleotides coding for nervous system growth factors ofinterest.

[0024] A. Neurotrophins

[0025] Known nervous system growth factors include nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),neurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6), ciliary neurotrophicfactor (CNTF), glial cell line-derived neurotrophic factor (GDNF), thefibroblast growth factor family (FGF's 1-15), leukemia inhibitory factor(LIF), certain members of the insulin-like growth factor family (e.g.,IGF-1), the neurturins, persephin, the bone morphogenic proteins (BMPs),the immunophilins, the transforming growth factor (TGF) family of growthfactors, the neuregulins, epidermal growth factor (EGF),platelet-derived growth factor (PDGF), and others. NGF and NT-3 inparticular have been tested with promising results in clinical trialsand animal studies (see, e.g., Hefti and Weiner, Ann Neurol., 20:275-281(1986); Tuszynki and Gage, Ann. Neurol., 30:625-636 (1991); Tuszynski,et al., Gene Therapy, 3:305-314 (1996) and Blesch and Tuszynski,Clin.Neurosci., 3:268-274 (1996)). Of the known nervous system growthfactors, NGF and NT-3 (for treatment of the Ch4 region, as in AD) arepreferred for use in the invention.

[0026] Human neurotrophins are preferred for use in therapy of humandisease according to the invention due to their relatively lowimmunogenicity as compared to allogenic growth factors. However, othernervous system growth factors are known which may also be suitable foruse in the invention with adequate testing of the kind described herein.

[0027] Coding polynucleotides for a number of human neurotrophins areknown, as are coding sequences for neurotrophins of other mammalianspecies (e.g., mouse, in which the coding sequence for NGF is highlyhomologous to the human coding sequence). For example, the codingsequence for hNGF is reported in GenBank at E03015 (Kazuo, et al.,Japanese Patent Application No. JP19911175976-A); for GDNF is reportedin GenBank at L190262 and L19063; genomic hNGF (with putative amino acidsequence) is reported in GenBank at HSBNGF (Ullrich, Nature, 303:821-825(1983)); the hNGF mRNA sequence is reported in GenBank at HSBNGFAC(Borsani, et al., Nucleic Acids Res., 18:4020 (1990); and the genomicnucleotide sequence of hNT3 is reported in GenBank at E07844 (Asae, etal., JP Patent Application No. 1993189770-A4). These references areincorporated herein to illustrate knowledge in the art concerningnucleotide and amino acid sequences for use in synthesis ofneurotrophins.

[0028] B. Recombinant Expression Vectors

[0029] The strategy for transferring genes into target cells in vivoincludes the following basic steps: (1) selection of an appropriatetransgene or transgenes whose expression is correlated with CNS diseaseor dysfunction; (2) selection and development of suitable and efficientvectors for gene transfer; (3) demonstration that in vivo transductionof target cells and transgene expression occurs stably and efficiently;(4) demonstration that the in vivo gene therapy procedure causes noserious deleterious effects; and (5) demonstration of a desiredphenotypic effect in the host animal.

[0030] Although other vectors may be used, preferred vectors for use inthe methods of the present invention are viral and non-viral vectors.The vector selected should meet the following criteria: 1) the vectormust be able to infect targeted cells and thus viral vectors having anappropriate host range must be selected; 2) the transferred gene shouldbe capable of persisting and being expressed in a cell for an extendedperiod of time (without causing cell death) for stable maintenance andexpression in the cell; and 3) the vector should do little, if any,damage to target cells.

[0031] Because adult mammalian brain cells are non-dividing, therecombinant expression vector chosen must be able to transfect and beexpressed in non-dividing cells. At present, vectors known to have thiscapability include DNA viruses such as adenoviruses, adeno-associatedvirus (AAV), and certain RNA viruses such as HIV-based lentiviruses,feline immunodeficiency virus (FIV) and equine immunodeficiency virus(EIV. Other vectors with this capability include herpes simplex virus(HSV). However, some of these viruses (e.g., AAV and HSV) can producetoxicity and/or immunogenicity. Recently, an HIV-based lentiviral vectorsystem has recently been developed which, like other retroviruses, caninsert a transgene into the nucleus of host cells (enhancing thestability of expression) but, unlike other retroviruses, can make theinsertion into the nucleus of non-dividing cells. Lentiviral vectorshave been shown to stably transfect brain cells after direct injection,and stably express a foreign transgene without detectable pathogenesisfrom viral proteins (see, Naldini, et al., Science, 272:263-267 (1996),the disclosure of which is incorporated by reference). Following theteachings of the researchers who first constructed the HIV-1 retroviralvector, those of ordinary skill in the art will be able to constructlentiviral vectors suitable for use in the methods of the invention (formore general reference concerning retrovirus construction, see, e.g.,Kriegler, Gene Transfer and Expression, A Laboratory Manual, W. FreemanCo. (NY 1990) and Murray, E J, ed., Methods in Molecular Biology, Vol.7, Humana Press (NJ 1991)).

[0032] Construction of vectors for recombinant expression of nervoussystem growth factors for use in the invention may be accomplished usingconventional techniques which do not require detailed explanation to oneof ordinary skill in the art. Specifics for construction of an HIV-1lentiviral vector are set forth in Example I. For further review, thoseof ordinary skill may wish to consult Maniatis et al., in MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (NY 1982).

[0033] Briefly, construction of recombinant expression vectors employsstandard ligation techniques. For analysis to confirm correct sequencesin vectors constructed, the ligation mixtures may be used to transform ahost cell and successful transformants selected by antibiotic resistancewhere appropriate. Vectors from the transformants are prepared, analyzedby restriction and/or sequenced by, for example, the method of Messing,et al., (Nucleic Acids Res., 9:309, 1981), the method of Maxam, et al.,(Methods in Enzymology, 65:499, 1980), or other suitable methods whichwill be known to those skilled in the art. Size separation of cleavedfragments is performed using conventional gel electrophoresis asdescribed, for example, by Maniatis, et al., (Molecular Cloning, pp.133-134, 1982).

[0034] Expression of a gene is controlled at the transcription,translation or post-translation levels. Transcription initiation is anearly and critical event in gene expression. This depends on thepromoter and enhancer sequences and is influenced by specific cellularfactors that interact with these sequences. The transcriptional unit ofmany prokaryotic genes consists of the promoter and in some casesenhancer or regulator elements (Banerji et al., Cell 27:299 (1981);Corden et al., Science 209:1406 (1980); and Breathnach and Chambon, Ann.Rev. Biochem. 50:349 (1981)). For retroviruses, control elementsinvolved in the replication of the retroviral genome reside in the longterminal repeat (LTR) (Weiss et al., eds., The molecular biology oftumor viruses: RNA tumor viruses, Cold Spring Harbor Laboratory, (NY1982)). Moloney murine leukemia virus (MLV) and Rous sarcoma virus (RSV)LTRs contain promoter and enhancer sequences (Jolly et al., NucleicAcids Res. 11:1855 (1983); Capecchi et al., In: Enhancer and eukaryoticgene expression, Gulzman and Shenk, eds., pp. 101-102, Cold SpringHarbor Laboratories (NY 1991). Other potent promoters include thosederived from cytomegalovirus (CMV) and other wild-type viral promoters.

[0035] Promoter and enhancer regions of a number of non-viral promotershave also been described (Schmidt et al., Nature 314:285 (1985); Rossiand de Crombrugghe, Proc. Natl. Acad. Sci. USA 84:5590-5594 (1987)).Methods for maintaining and increasing expression of transgenes inquiescent cells include the use of promoters including collagen type I(1 and 2) (Prockop and Kivirikko, N. Eng. J. Med. 311:376 (1984); Smithand Niles, Biochem. 19:1820 (1980); de Wet et al., J. Biol. Chem.,258:14385 (1983)), SV40 and LTR promoters.

[0036] In addition to using viral and non-viral promoters to drivetransgene expression, an enhancer sequence may be used to increase thelevel of transgene expression. Enhancers can increase thetranscriptional activity not only of their native gene but also of someforeign genes (Armelor, Proc. Natl. Acad. Sci. USA 70:2702 (1973)). Forexample, in the present invention collagen enhancer sequences are usedwith the collagen promoter 2(I) to increase transgene expression. Inaddition, the enhancer element found in SV40 viruses may be used toincrease transgene expression. This enhancer sequence consists of a 72base pair repeat as described by Gruss et al., Proc. Natl. Acad. Sci.USA 78: 943 (1981); Benoist and Chambon, Nature 290:304 (1981), andFromm and Berg, J. Mol. Appl. Genetics, 1:457 (1982), all of which areincorporated by reference herein. This repeat sequence can increase thetranscription of many different viral and cellular genes when it ispresent in series with various promoters (Moreau et al., Nucleic AcidsRes. 9:6047 (1981).

[0037] Transgene expression may also be increased for long term stableexpression using cytokines to modulate promoter activity. Severalcytokines have been reported to modulate the expression of transgenefrom collagen 2(I) and LTR promoters (Chua et al., connective TissueRes., 25:161-170 (1990); Elias et al., Annals N.Y. Acad. Sci.,580:233-244 (1990)); Seliger et al., J. Immunol. 141:2138-2144 (1988)and Seliger et al., J. Virology 62:619-621 (1988)). For example,transforming growth factor (TGF), interleukin (IL)-1, and interferon(INF) down regulate the expression of transgenes driven by variouspromoters such as LTR. Tumor necrosis factor (TNF) and TGF1 up regulate,and may be used to control, expression of transgenes driven by apromoter. Other cytokines that may prove useful include basic fibroblastgrowth factor (bFGF) and epidermal growth factor (EGF).

[0038] Collagen promoter with the collagen enhancer sequence (Coll(E))can also be used to increase transgene expression by suppressing furtherany immune response to the vector which may be generated in a treatedbrain notwithstanding its immune-protected status. In addition,anti-inflammatory agents including steroids, for example dexamethasone,may be administered to the treated host immediately after vectorcomposition delivery and continued, preferably, until anycytokine-mediated inflammatory response subsides. An immunosuppressionagent such as cyclosporin may also be administered to reduce theproduction of interferons, which downregulates LTR promoter and Coll(E)promoter-enhancer, and reduces transgene expression.

[0039] C. Pharmaceutical Preparations

[0040] To form a neurotrophic composition for use in the invention,neurotrophin encoding expression vectors (including, without limitation,viral and non-viral vectors) may be placed into a pharmaceuticallyacceptable suspension, solution or emulsion. Suitable mediums includesaline and liposomal preparations.

[0041] More specifically, pharmaceutically acceptable carriers mayinclude sterile aqueous of non-aqueous solutions, suspensions, andemulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like.

[0042] Preservatives and other additives may also be present such as,for example, antimicrobials, antioxidants, chelating agents, and inertgases and the like. Further, a composition of neurotrophin transgenesmay be lyophilized using means well known in the art, for subsequentreconstitution and use according to the invention.

[0043] A colloidal dispersion system may also be used for targeted genedelivery. Colloidal dispersion systems include macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.Liposomes are artificial membrane vesicles which are useful as deliveryvehicles in vitro and in vivo. It has been shown that large unilamellarvesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate asubstantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA and intact virions can be encapsulated within theaqueous interior and be delivered to cells in a biologically active form(Fraley, et al., Trends Biochem. Sci., 6:77, 1981). In addition tomammalian cells, liposomes have been used for delivery of operativelyencoding transgenes in plant, yeast and bacterial cells. In order for aliposome to be an efficient gene transfer vehicle, the followingcharacteristics should be present: (1) encapsulation of the genesencoding the antisense polynucleotides at high efficiency while notcompromising their biological activity; (2) preferential and substantialbinding to a target cell in comparison to non-target cells; (3) deliveryof the aqueous contents of the vesicle to the target cell cytoplasm athigh efficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

[0044] The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

[0045] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0046] The targeting of liposomes can be classified based on anatomicaland mechanistic factors. Anatomical classification is based on the levelof selectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0047] The surface of the targeted gene delivery system may be modifiedin a variety of ways. In the case of a liposomal targeted deliverysystem, lipid groups can be incorporated into the lipid bilayer of theliposome in order to maintain the targeting ligand in stable associationwith the liposomal bilayer. Various linking groups can be used forjoining the lipid chains to the targeting ligand.

[0048] IV. Methods for Delivery of Vector Composition

[0049] Following the protocol defined by the invention, direct deliveryof a neurotrophic composition may be achieved by means familiar to thoseof skill in the art, including microinjection through a surgicalincision (see, e.g., Capecchi, Cell, 22:479-488 (1980)); electropotation(see, e.g., Andreason and Evans, Biotechniques, 6:650-660 (1988));infusion, chemical complexation with a targeting molecule orco-precipitant (e.g., liposome, calcium), and microparticle bombardmentof the target tissue (Tang, et al., Nature, 356:152-154 (1992)).

[0050] As used in this disclosure, “unit dosage” refers generally to theconcentration of neurotrophin/ml of neurotrophic composition. For viralvectors, the neurotrophin concentration is defined by the number ofviral particles/ml of neurotrophic composition. Optimally, for deliveryof neurotrophin using a viral expression vector, each unit dosage ofneurotrophin will comprise 2.5 to 25 μl of a neurotrophic composition,wherein the composition includes a viral expression vector in apharmaceutically acceptable fluid and provides from 10¹⁰ up to 10¹⁵ NGFexpressing viral particles per ml of neurotrophic composition.

[0051] The neurotrophic composition is delivered to each delivery cellsite in the target tissue by microinjection, infusion, scrape loading,electroporation or other means suitable to directly deliver thecomposition directly into the delivery site tissue through a surgicalsincision. The delivery is accomplished slowly, such as over a period ofabout 5-10 minutes (depending on the total volume of neurotrophiccomposition to be delivered).

[0052] Those of skill in the art will appreciate that the directdelivery method employed by the invention obviates a limiting riskfactor associated with in vivo gene therapy; to wit, the potential fortransfection of non-targeted cells with the vector carrying the NGFencoding transgene. In the invention, delivery is direct and thedelivery sites are chosen so diffusion of secreted NGF takes place overa controlled and predetermined region of the brain to optimize contactwith targeted neurons, while minimizing contact with non-targeted cells.

[0053] Startlingly, in primates, viral vectors with an operableneurotrophin encoding transgene have been shown to express humanneurotrophin after delivery to the brain and to the CNS for up to 12months (Example VIII). As such, the invention provides a chronicallyavailable source for neurotrophin in the brain.

[0054] V. Animal Models and Clinical Evaluation

[0055] In non-human primate subjects, the process of aging simulates theneurological changes in the brain experienced in aging humans (ExampleII). An non-aged animal model that models Parkinson's Disease with ahigh degree of integrity is 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) treated monkeys (see, e.g., Kordower, et al., Exp. Neurology,160:1-16 (1999). Such treatment results in extensive degeneration ofdopaminergic neurons in the substantia nigra, with concomitantbehavioral modification and motor deficits (Example V). Datademonstrating the use and efficacy of the method of the invention inaged non-human primates is provided in Examples III and IV; and, in MPTPtreated animals, in Examples V and VI. The absence of lentiviralimmunogenicity in treated animals is confirmed in Example VII.

[0056] Clinical evaluation and monitoring of treatment can be performedusing the in vivo imaging techniques described above as well as throughbiopsy and histological analysis of treated tissue. In the latterrespect, neuronal numbers can be quantified in a tissue sample withrespect to, for example, TH immunoreactivity, anti-neurotrophin antibody(for immunoassay of secreted neurotrophin) or NGF-receptor (p75), andcholine acetyltransferase (ChAT) for labeling of neurons. A sampleprotocol for in vitro histological analysis of treated and controltissue samples is described in the Example II.

[0057] The invention having been fully described, examples illustratingits practice are set forth below. These examples should not, however, beconsidered to limit the scope of the invention, which is defined by theappended claims. Those of ordinary skill in the art will appreciate thatwhile the Examples illustrate an ex vivo application of the invention,the results achieved will be accessible through in vivo delivery of thenervous system growth factor encoding transgenes described, as taughtherein, with in vivo gene delivery sites and direct delivery meanssubstituted for the grafting sites and grafting methods discussed in theExamples.

[0058] In the examples, the abbreviation “min.” refers to minutes, “hrs”and “h” refer to hours, and measurement units (such as “ml”) arereferred to by standard abbreviations. All printed materials cited areincorporated herein by reference.

EXAMPLE I Construction of GDNF Expressing Lentiviral Vector

[0059] The cDNA coding for a nuclear-localized β-galactosidase (LacZ)and the human GDNF containing a Kozak consensus sequence (a 636-bpfragment: position 1 to 151 and 1 to 485; GenBank accession numbersL19062 and L19063) were cloned in the SIN-W-PGK transfer vector (R.Zufferey, et al., J. Virol. 73, 2886 (1999), incorporated herein byreference). The packaging construct and vesicular stomatis virus Gprotein (VSV-G) envelope used in this study were the PCMVDR8.92,PRSV-Rev, and the PMD.6 plasmids described previously (see, R. Zufferey,D. Nagy, R. J. Mandel, L. Naldini, D. Trono, Nature Biotechnol. 15, 871(1997); A. F. Hottinger, M. Azzouz, N. Déglon, P. Gebischer, A. D. Zurn,J. Neurosci. 20, 5587 (2000), incorporated herein by reference). Theviral particles were produced in 293T cells as previously described.

[0060] The titers (3 to 5×108 TU/ml) of the concentrated LacZ-expressingviruses (200,000 and 250,000 ng p24/ml in experiment 1 and 450,000 ngp24/ml in experiment 2) were determined on 293T cells. TheGDNF-expressing viral stocks were normalized for viral particles contentusing p24 antigen measurement.

EXAMPLE II Transgene Expression and Anterograde Transport of GDNFExpression Product Within the Aged Primate Brain

[0061] Non-lesioned aged monkeys that model PD like neurodegeneration(17) display a slow progressive loss of dopamine within the striatum andtyrosine hydroxylase (TH) within the substantia nigra without frankcellular degeneration. Eight aged (approximately 25 years old) femalerhesus monkeys received injections of lentiviral vectors encodinggalactosidase (lenti-Gal; n=4) or GDNF (lenti-GDNF; n=4) targeted forthe striatum and substantia nigra and were killed 3 months later.

[0062] Under MRI guidance, each monkey received six stereotaxicinjections of lenti-Gal or lenti-GDNF bilaterally into the caudatenucleus, putamen, and substantia nigra. Injections were made into thehead of the caudate nucleus (10 μl), body of the caudate nucleus (5 μl),anterior putamen (10 μl), commissural putamen (10 μl), postcommissuralputamen (5 μl), and substantia nigra (5 μl). Injections were madethrough a 10-μl Hamilton syringe connected to a pump at a rate of 0.5μl/min.

[0063] During the injection, the needle was raised 1 to 2 mm to betterdisperse the lentivirus through the intended target. The needle was leftin place for an additional 3 min to allow the injectate to diffuse fromthe needle tip. The left side was injected 6 weeks before the right.During the first surgical session, there was a technical failure withthe virus aggregating in the needle, which prevented its injection intothe brain. This was confirmed at postmortem examination usingGDNF-immunohistochemistry and Gal histochemistry. Thus, the left sideserved as an additional control for the right side. Postmortem, all GDNFinjections were localized to the caudate nucleus, putamen, andsupranigral regions, as revealed by standard staining procedures.

[0064] All aged monkeys receiving lenti-GDNF displayed robust GDNFimmunoreactivity within the right striatum (FIG. 1A) and substantianigra (FIG. 1C). In contrast, no monkeys receiving lenti-Gal displayedspecific GDNF immunoreactivity in the right striatum (FIG. 1B). Rather,these monkeys displayed robust expression of Gal similar to thatreported previously. In lenti-GDNF-treated animals, GDNFimmunoreactivity within the striatum was extremely dense and distributedthroughout the neuropil (FIG. 1). When the primary antibodyconcentration was decreased to one-tenth of the standard, the intensestriatal neuropil staining was diminished, and GDNF-immunoreactiveperikarya were easily seen. Numerous GDNF-immunoreactive perikarya werealso seen within the substantia nigra of lenti-GDNF-injected monkeys.

[0065] Within the striatum and substantia nigra, Niss1-stained sectionsrevealed normal striatal cytoarchitecture without significantcytotoxicity. Macrophages were occasionally observed within the needletracts. Gliosis was similar across treatment groups and was principallyconfined to the regions immediately surrounding the needle tracts.Lenti-GDNF injections resulted in marked anterograde transport of thetrophic factor. Intense GDNF immunoreactivity was observed within fibersof the globus pallidus (FIG. 1D) and substantia nigra pars reticulata(FIG. 1E) after striatal injections. GDNF-containing fibers emanatingfrom putaminal injection sites were seen coursing medially toward andinto the globus pallidus (FIG. 1D). These staining patterns were clearlydistinct from the injection site and respected the boundaries of thestriatal target structures.

[0066] In contrast, anterograde transport of Gal was not observed inlenti-Gal monkeys. This suggests that secreted GDNF, and not the virusper se, was anterogradely transported.

EXAMPLE III DOPA Uptake Following GDNF Treatment in Aged Animals

[0067] Aged monkeys underwent fluorodopa (FD) positron emissiontomography (PET) before surgery and again just before being killed.Before treatment, all monkeys displayed symmetrical FD uptake in thecaudate and putamen bilaterally (ratio: 1.02±0.02) (FIGS. 2A and 2B,left). Similarly, there was symmetrical (4% difference) FD uptake in alllenti-Gal-treated monkeys after lentivirus injections (FIG. 2A, right).

[0068] In contrast, FD uptake was significantly asymmetrical (27%) inlenti-GDNF-treated monkeys with greater uptake on the side of the GDNFexpression (P<0.007; FIG. 2B, right). With respect to absolute values,lenti-Gal animals displayed a trend toward reduced FD uptake aftertreatment relative to baseline levels (P=0.06). Qualitatively, three offour lenti-GDNF-treated monkeys displayed clear increases in FD uptakeon the treated side.

[0069] Within the striatum, lentiviral delivery of GDNF increased anumber of markers of dopaminergic function. Optical density measurementswere performed to assess the relative intensity of TH staining withinthe caudate nucleus and putamen (FIG. 3, A and B).

[0070] On the left side where there was no lenti-GDNF expression, theintensity of TH immunoreactivity within the caudate nucleus and putamenwas similar between groups (FIG. 3, A and B). In contrast, significantincreases in optical density measures of TH immunoreactivity were seenin the right striatum of lenti-GDNF-infused monkeys (FIG. 3A) relativeto lenti-Gal-treated animals (FIG. 3B) or the contralateral side (FIG.3A). In this regard, there was a 44.1% and a 38.9% increase in opticaldensity measures of TH immunoreactivity within the caudate nucleus andputamen, respectively (FIG. 4D). At the time of death, tissue puncheswere taken throughout the caudate nucleus and putamen of all monkeys.Relative to lenti-Gal-treated animals, measurement of dopamine (DA) andhomovanillic acid (HVA) revealed significant increases in the rightcaudate nucleus (140% DA, P<0.001; 207% HVA, P<0.001) and putamen (47.2%DA, P<0.05; 128% HVA, P<0.01) in lenti-GDNF-treated aged monkeys (FIG.4, E and F).

EXAMPLE IV Neuron Generation in Aged Animals

[0071] Lentiviral delivery of GDNF to aged monkeys resulted in anincrease in the number of TH-immunoreactive neurons within thesubstantia nigra (FIG. 3, C and D). Regardless of the extent of GDNFimmunoreactivity within the midbrain, the organization ofTH-immunoreactive neurons was similar in all animals, and these neuronswere not observed in ectopic locations within this locus.

[0072] Stereological counts revealed an 85% increase in the number ofTH-immunoreactive nigral neurons on the side receiving lentivirallydelivered GDNF (FIG. 4A) relative to lenti-Gal-treated animals. On theside (left) that did not display GDNF immunoreactivity,lenti-GDNF-treated animals contained 76,929±4918 TH-immunoreactiveneurons. This is similar to what was seen in lenti-Gal-infused animals(68,543±5519). Whereas lenti-Gal-infused monkeys contained 63,738±6094TH-immunoreactive nigral neurons in the right side, lenti-GDNF-treatedmonkeys contained 118,170±8631 TH-immunoreactive nigral neurons in thishemisphere (P<0.001).

[0073] A similar pattern was seen when the volume of TH-immunoreactivesubstantia nigra neurons was quantified (FIG. 4B). TH-immunoreactiveneurons from lenti-Gal- and lenti-GDNF-treated monkeys were similar insize in the left nigra where there was no GDNF expression (11,147.5±351μm3 and 11,458.7±379 μm3, respectively). In contrast, a 35% increase inneuronal volume was seen on the GDNF-rich right side inlenti-GDNF-injected aged monkeys (lenti-Gal 10,707.5±333 μm3; lenti-GDNF16,653.7±1240 μm3; P<0.001).

[0074] Although stereological counts of TH mRNA-containing neurons werenot performed, there was an obvious increase in the number of THmRNA-containing neurons within the right substantia nigra inlenti-GDNF-treated monkeys (FIG. 3E) compared with lenti-Gal-containinganimals (FIG. 3F). With regard to the relative levels of TH mRNAexpression within individual nigral neurons, the pattern of results wassimilar to that observed with TH-immunoreactive neuronal number andvolume (FIG. 4C). On the left side, the optical density of TH mRNAwithin nigral neurons was similar between lenti-Gal- andlenti-GDNF-treated monkeys (78.28±2.78 and 80.58±2.5, respectively). Incontrast, there was a significant (21.5%) increase in the opticaldensity for TH mRNA in lenti-GDNF-treated monkeys (98.3±1.5) relative tolenti-Gal-treated monkeys (77.2±2.3) on the right side (P<0.01).

EXAMPLE V Functional Recovery in Chemically Impaired Animals

[0075] For a second model of neurodegenerative changes similar to thoseoccurring in PD, young adult monkeys received unilateral intracarotidinjections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toinduce extensive nigrostriatal degeneration, resulting in a behavioralsyndrome characterized by robust motor deficits.

[0076] In the second experiment, 20 young adult rhesus were initiallytrained 3 days per week until asymptotic performance was achieved on ahand-reach task in which the time to pick up food treats out of recessedwells was measured (16, 24). Each experimental day, monkeys received 10trials per hand. Once per week, monkeys were also evaluated on amodified parkinsonian clinical rating scale (CRS). All monkeys thenreceived an injection of 3 mg MPTP-HCl into the right carotid artery,initiating a parkinsonian state.

[0077] One week later, monkeys were evaluated on the CRS. Only monkeysdisplaying severe hemiparkinsonism with the classic crooked arm postureand dragging leg on the left side continued in the study (n=10). Monkeyswith this behavioral phenotype generally display the most severe lesionsneuroanatomically and do not display spontaneous recovery behaviorally.

[0078] On the basis of CRS scores, monkeys were matched into two groupsof five monkeys, which received on that day lenti-Gal or lenti-GDNFtreatment. Using magnetic resonance imaging (MRI) guidance, all monkeyswere given lentivirus injections into the caudate nucleus (n=2), putamen(n=3), and substantia nigra (n=1) on the right side using the sameinjection parameters as in experiment 1. One week later, monkeys beganretesting on the hand-reach task three times per week for 3 weeks permonth.

[0079] For statistical analyses, the times for an individual week werecombined into a single score. During the weeks of hand-reach testing,monkeys were also scored once per week on the CRS. Individuals blindedto the experimental treatment performed all behavioral assessments.

[0080] Before MPTP treatment, all young adult monkeys scored 0 on theCRS. After MPTP, but before lentivirus injection, monkeys in thelenti-GDNF and lenti-Gal groups averaged 10.4±0.07 and 10.6±0.6,respectively, on the CRS (P>0.05). After lentivirus treatment,significant differences in CRS scores were seen between the two groups(Kolmogorov-Smirnov test, P<0.0001; FIG. 5A). CRS scores of monkeysreceiving lenti-Gal did not change over the 3-month period aftertreatment. In contrast, CRS scores of monkeys receiving lenti-GDNFsignificantly diminished during the 3-month period after treatment.Scores began to decrease in the first month after lenti-GDNF treatment.However, statistically significant differences between lenti-GDNF andlenti-Gal were only discerned at posttreatment observations 6, 7, 8, and9 (Kolmogorov-Smirnov test, P <0.04 for each comparison).

[0081] Lenti-GDNF-treated animals also improved performance on theoperant hand-reach task. Under the conditions before MPTPadministration, animals in both groups performed this task with similarspeed (FIG. 5B). For the “unaffected” right hand, no differences inmotor function were discerned for either group relative to performancebefore MPTP administration or to each other (P>0.05). In contrast,performance with the left hand was significantly improved inlenti-GDNF-treated animals relative to controls (P<0.05).

[0082] After MPTP, all lenti-Gal-treated animals were severely impaired,with monkeys often not performing at all, or requiring more than themaximally allowed 30 s. In contrast, three of the four lenti-GDNFmonkeys performed the task with the left hand at near-normal levels,whereas one lenti-GDNF-treated monkey was impaired and performed thistask in a manner similar to the lenti-Gal-treated animals. Betweengroups, significant differences in performance were discerned onposttreatment tests 4, 6, 7, 8, and 9 (P<0.05 for each comparison).

EXAMPLE VI Neuron Generation in Chemically Impaired Animals

[0083] All monkeys underwent FD PET scans. Qualitatively, alllenti-Gal-treated monkeys displayed pronounced FD uptake in the leftstriatum and a comprehensive loss of FD uptake on the right side (FIG.2C). In contrast, two of four lenti-GDNF-treated animals displayedrobust and symmetrical FD uptake on both sides (FIG. 2D). The remainingtwo lenti-GDNF monkeys displayed reduced FD uptake on the right side,but with Ki values 50 to 100% greater than lenti-Gal controls (FIG. 2).Quantitatively, no differences in FD uptake were observed between groupswithin the left striatum (P>0.05). In contrast, there was a significant(>300%) increase in FD uptake in lenti-GDNF-treated animals in the rightstriatum relative to lenti-Gal-treated animals (P<0.05). When the rightstriatum was subdivided, significant increases in FD uptake were onlyseen within the putamen of lenti-GDNF-treated animals (P<0.05).

[0084] After death, a strong GDNF-immunoreactive signal was seen in thecaudate nucleus, putamen, and substantia nigra of alllenti-GDNF-treated, but none of the lenti-Gal-treated animals. Theintensity and distribution of GDNF immunoreactivity wasindistinguishable from what was observed in aged monkeys (see FIG. 1).

[0085] All lenti-Gal-treated monkeys displayed a comprehensive loss ofTH immunoreactivity within the striatum on the side ipsilateral to theMPTP injection (FIG. 6A). In contrast, all lenti-GDNF-treated monkeysdisplayed enhanced striatal TH immunoreactivity relative to Gal controls(FIG. 6B).

[0086] There was variability in the degree of striatal THimmunoreactivity in lenti-GDNF-treated animals and that variability wasassociated with the degree of functional recovery seen on the hand-reachtask. Two lenti-GDNF-treated monkeys displayed dense TH immunoreactivitythroughout the rostrocaudal extent of the striatum (FIG. 6B). In thesemonkeys, the intensity of the TH immunoreactivity was greater than thatobserved on the intact side. These two animals displayed the bestfunctional recovery.

[0087] A third lenti-GDNF-treated monkey also displayed robustfunctional recovery on the hand-reach task. However, the enhancedstriatal TH immunoreactivity in this animal was limited to thepost-commissural putamen. The fourth lenti-GDNF-treated monkey did notrecover on the hand-reach task. Although putaminal TH immunoreactivityin this animal was still greater than controls, the degree ofinnervation was sparse and restricted to the medial post-commissuralputamen.

[0088] Lenti-GDNF treatment enhanced the expression of TH-immunoreactivefibers throughout the nigrostriatal pathway. Unlike what was observed inaged monkeys, however, some TH-immunoreactive fibers in the striatumdisplayed a morphology characteristic of both degenerating andregenerating fibers. Large, thickened fibers could be seen coursing inan irregular fashion in these animals. Rostrally, these fibers appeareddisorganized at times, with a more normal organization seen morecaudally. TH-immunoreactive sprouting was also seen in the globuspallidus (FIG. 6, G and H), substantia innominata (FIG. 6, A and B), andlateral septum. These novel staining patterns were not immunoreactivefor dopamine-hydroxylase confirming the dopaminergic phenotype of thisresponse. Quantitatively, lenti-Gal-treated monkeys displayedsignificant decreases in the optical density of TH-immunoreactive fiberswithin the right caudate nucleus (71.5%; P<0.006; FIG. 7D) and putamen(74.3% P<0.0007; FIG. 7D) relative to the intact side. When analyzed asa group, TH optical density in the right caudate nucleus and putamen oflenti-GDNF-treated monkeys was significantly greater than that seen inlenti-Gal-treated monkeys (P<0.001 for both) and was similar to thatseen on the intact side of these animals (P>0.05 for both).

[0089] All lenti-Gal-treated monkeys displayed a dramatic loss ofTH-immunoreactive neurons within the substantia nigra on the sideipsilateral to the MPTP injection (FIG. 7A). In contrast, the nigra fromall four of the lenti-GDNF-treated displayed complete neuroprotection(FIG. 7A), regardless of the degree of functional recovery. Inlenti-Gal-treated monkeys, intracarotid injections of MPTP resulted inan 89% decrease in the number (FIG. 7A), and an 81.6% decrease in thedensity, of TH-immunoreactive nigral neurons on the side ipsilateral tothe toxin injection (P<0.001).

[0090] In contrast, lenti-GDNF-treated monkeys displayed 32% moreTH-immunoreactive nigral neurons (P<0.001) and an 11% increase inTH-immunoreactive neuronal density (P<0.05) relative to the intact side.In lenti-Gal-treated animals, MPTP significantly reduced (32%) thevolume of residual TH-immunoreactive nigral neurons on the lesion siderelative to the intact side (P<0.001; FIG. 7B). The volume ofTH-immunoreactive neurons in lenti-GDNF-treated animals wassignificantly larger (44.3%) on the lesioned side relative to the intactside (P<0.001).

[0091] Finally, the optical density of TH mRNA was quantifiedbilaterally in all animals (FIG. 7C). In lenti-Gal-treated animals,there was a significant decrease (24.0%) in the relative optical densityof TH mRNA within residual neurons on the MPTP-lesioned side relative tothe intact side (P<0.03). In contrast, lenti-GDNF-treated animalsdisplayed a significant increase (41.7%) in relative optical density ofTH mRNA relative to the intact side or lenti-Gal-treated animals(P<0.001).

EXAMPLE VII Absence of Lentiviral Immunoreactivity in Treated Animals

[0092] Sections from all monkeys were stained for CD45, CD3, and CD8markers to assess the immune response after lentiviral vector injection.These antibodies are markers for activated microglia, T cells, andleukocytes including lymphocytes, monocytes, granulocytes, eosinophils,and thymocytes. Staining for these immune markers was weak, and oftenabsent, in these animals. Mild staining for CD45 and CD8 was seen in twoanimals. Some CD45-immunoreactive cells displayed a microglialmorphology. Other monkeys displayed virtually no immunoreactivity evenin sections containing needle tracts.

EXAMPLE VIII Long Term GDNF Expression in Treated Animals

[0093] Two additional intact young adult rhesus monkeys receivedlenti-GDNF injections into the right caudate and putamen and the leftsubstantia nigra using the same injection protocol utilized in ExampleVI. These animals were killed 8 months later and were evaluated byimmunohistochemistry and enzyme-linked immunosorbent assay (ELISA) forlong-term gene expression.

[0094] Robust GDNF immunoreactivity was seen in the right caudate, rightputamen, and left ventral midbrain in both animals. In the rightsubstantia nigra, many GDNF-immunoreactive neurons were seen. Thislabeling represents retrograde transport of GDNF after injections oflenti-GDNF into the right striatum. Further, dense GDNF-immunoreactivefiber staining, representing anterograde transport of the trophicfactor, was seen within the right substantia nigra pars reticulata.Tissue punches taken at the time of death revealed significant levels ofGDNF produced by striatal cells 8 months after lenti-GDNF injections.

[0095] On the side without a striatal injection, 0.130±0.062 and0.131±0.060 ng/mg protein of GDNF were seen in the caudate nucleus andputamen, respectively. Significantly higher GDNF levels were observedwithin the caudate nucleus (2.25±0.312 ng/mg protein; P<0.001) andputamen (3.5±0.582 ng/mg protein; P<0.001) on the lenti-GDNF-injectedside.

The invention claimed is:
 1. A method for delivery of a therapeuticneurotrophin to targeted defective, diseased or damaged neurons in themammalian brain, the method comprising delivering a neurotrophiccomposition, comprising a neurotrophin encoding lentiviral expressionvector, into one or more delivery sites within a region of the braincontaining targeted neurons; wherein the neurotrophin is expressed in,or within 500 μm from, a targeted cell, and no more than about 10 mmfrom another delivery site; and wherein further contact with theneurotrophin ameliorates the defect, disease or damage.
 2. The methodaccording to claim 1, wherein the region of the brain containing thetargeted neurons is the substantia nigra.
 3. The method according toclaim 2, wherein the targeted neurons are dopaminergic neurons.
 4. Themethod according to claim 1, wherein the viral expression vector isHIV-1.
 5. The method according to claim 1, wherein the neurotrophiccomposition is a fluid having a concentration of neurotrophin encodinglentiviral particles in the range from 10¹⁰ to 10¹⁵ particles per ml ofneurotrophic composition.
 6. The method according to claim 5, whereinfrom 2.5 μl to 25 μl of the neurotrophic composition is delivered toeach delivery site.
 7. The method according to claim 1, wherein thetreated mammal is a human and the transgene encodes a humanneurotrophin.
 8. The method according to claim 7, wherein theneurotrophin is human glial cell-derived neurotrophic factor (GDNF). 9.The method according to claim 7, wherein the human is suffering fromParkinson's disease, and the disease is ameliorated by stimulation ofgrowth of dopaminergic neurons.
 10. The method according to claim 9,wherein the disease is ameliorated by reversal of deficits in motorfunction associated with the Parkinson's disease.
 11. The methodaccording to claim 7, wherein the human is suffering from Alzheimer'sdisease, and the disease is ameliorated by stimulation of growth ofcholinergic neurons.
 12. The method according to claim 11, wherein thedisease is ameliorated by improvement of cognitive function whoseimpairment was associated with Alzheimer's disease.